Cryptococcus neoformans is a neurotropic pathogen that causes fatal meningoencephalitis primarily in individuals with T-cell deficiency such as the AIDS patients. However, the fungus also causes infection in otherwise normal patients at a low frequency. The disease is 100% fatal unless treated and even with the most effective antimycotic agents, the fatality rate is about 25%. Current estimates of annual deaths due to cryptococcosis world-wide is close to 700,000. C. neoformans is commonly found in the human environment world-wide. In previous years, we discovered that C. neoformans yeast cells invaded the brain by crossing the blood-brain barrier transcellularly. We also, reported that the product of CPS1 gene that plays an important role in association and trversal of C. neoformans yeast cells across the blood-brain barrier (BBB)is hyaluronic acid which is located at the base of the extracellular polysaccharide that surrounds the yeast cells. We also showed, in collaboration with Dr. Jong's group, that protein kinase C-alpha activation is required for the yeast to cross the BBB and CD44 is involved in the association of the yeast cells with the brain microvasular endothelial cells. In order to gain better insight into the invasion process, we have taken a genetic approach during 2008-2009 period. During 2009-2010, in collaboration with Dr. Kim at Johns Hopkins University, we found that the host cell Rac1 GTPase contributes to C. neoformans traversal of the blood-brain barrier and pharmacological inhibition of Rac1 was efficient in inhibiting C. neoformans traversal of the blood-brain barrier, the essential step for the development of C. neoformans meningoencephalitis. This was shown by our demonstrations that (a) Rac1 activation occurs in response to C. neoformans in human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier, (b) inhibition of Rac1 was efficient in prevention of HBMEC traversal for both encapsulated and acapsular strains of C. neoformans, and (c) pharmacological inhibition of Rac1 significantly decreased C. neoformans penetration into the brain. These findings demonstrate that targeting host cell signaling molecules essential to C. neoformans traversal of the blood-brain barrier, as shown here with Rac1 GTPase, provides a novel approach to prevention of C. neoformans meningoencephalitis. During 2010-2011,in collaboration with Dr. Jong at the University of Southern California,we found several lines of evidence indicating that C. neoformans invasion is mediated through the endocytic pathway via lipid rafts. Human CD44 molecules from lipid rafts can directly interact with hyaluronic acid, the fungal ligand. Bikunin, which perturbs CD44 function in the lipid raft, can block fungal adhesion and invasion of HBMEC. The lipid raft marker, ganglioside GM1, colocalizes with CD44 on the plasma membrane, and fungal cells can adhere to the host cells where GM1 is enriched. The results suggest the fungal entry is taking place on the lipid rafts. Upon fungal engagement, GM1 was internalized through vesicular structures to the nuclear membrane. This endocytic redistribution process can be perturbed by cytochalasin D, nocodazole, and anti-DYRK3 siRNA. Concomitantly, the knockdown of DYRK3 significantly reduces fungal invasion across the HBMEC monolayer in vitro. Our data demonstrated that the lipid-raft dependent endocytosis process mediates C. neoformans internalization into HBMEC, in which the hosts CD44 protein, cytoskeleton, and intracellular kinase-DYRK3 are involved.During 2011-2012, we investigated the cryptococcal factors that can activate the host signaling pathways that enhance C. neoformans crossing of the blood-brain barrier by using the HBMEC and found that phospholipase secreted by yeast cells of C. neoformans first activates RhoGTPases followed by PKC-alpha, FAK and ezrin.